Anders Buchard

Multiplex PCR and minisequencing of SNPs— a model with 35 Y chromosome SNPs

We have developed a robust single nucleotide polymorphism (SNPs) typing assay with co-amplification of 25 DNA-fragments and the detection of 35 human Y chromosome SNPs. The sizes of the PCR products ranged from 79 to 186 base pairs. PCR primers were designed to have a theoretical Tm of 60 +/- 5 degrees C at a salt concentration of 180 mM. The sizes of the primers ranged from 19 to 34 nucleotides. The concentration of amplification primers was adjusted to obtain balanced amounts of PCR products in 8mM MgCl2. For routine purposes, 1 ng of genomic DNA was amplified and the lower limit was approximately 100 pg DNA. The minisequencing reactions were performed simultaneously for all 35 SNPs with fluorescently labelled dideoxynucleotides. The size of the minisequencing primers ranged from 19 to 106 nucleotides. The minisequencing reactions were analysed by capillary electrophoresis and multicolour fluorescence detection. Female DNA did not influence the results of Y chromosome SNP typing when added in concentrations more than 300 times the concentrations of male DNA. The frequencies of the 35 SNPs were determined in 194 male Danes. The gene diversity of the SNPs ranged from 0.01 to 0.5.

Non-uniform phenotyping of D12S391 resolved by second generation sequencing

Non-uniform phenotyping of five case work samples were observed in the D12S391 locus. The samples were typed at least twice with the AmpFℓSTR NGM SElect PCR Amplification Kit and different alleles were called with GeneMapper ID-X in the different experiments. Detailed analyses of the electropherograms suggested that the individuals were heterozygous with two alleles that differed in size by one nucleotide. This was confirmed by amplifying the samples with the PowerPlex ESX 17 system. D12S391 is a complex STR with variable numbers of AGAT and AGAC repeats. Second generation sequencing revealed that separation of two alleles differing by one nucleotide in length was poor if the number of AGAT repeats in the short allele was higher than in the long allele. A total of 45 individuals with microvariants or off-ladder alleles in D12S391 were sequenced. Thirty different alleles were detected and sixteen of these were not previously reported.

Forensic and population genetic analyses of Danes, Greenlanders and Somalis typed with the Yfiler® Plus PCR amplification kit.

Recently, the Yfiler® Plus PCR Amplification Kit (Yfiler® Plus, Thermo Fisher Scientific, Waltham, MA, USA) was introduced. Yfiler® Plus amplifies 27 Y-chromosomal short tandem repeat loci (Y-STRs) and adds ten new Y-STRs to those analysed with the commonly used AmpFlSTR® Yfiler® PCR Amplification Kit (Yfiler®, Thermo Fisher Scientific, Waltham, MA, USA). Seven of the new Y-STRs are rapidly mutating Y-STRs (RM Y-STRs). In this study, 551 male individuals from Denmark, Greenland and Somalia were typed with Yfiler® Plus. The results were compared to those obtained with Yfiler® in the same individuals. Forensic and population genetic parameters were estimated for Yfiler® Plus. Yfiler® Plus had a higher power of discrimination than Yfiler® in all three populations. Compared to Yfiler®, Yfiler® Plus offers increased power of discrimination, which is obviously an advantage in crime case investigations. However, the inclusion of seven RM Y-STRs in Yfiler® Plus makes it less attractive for relationship testing because of the relatively high combined mutation rate, approximately 15%.

Postmortem Blood Concentrations of R‐ and S‐Enantiomers of Methadone and EDDP in Drug Users: Influence of Co‐Medication and P‐glycoprotein Genotype

Abstract:  We investigated toxicological and pharmacogenetic factors that could influence methadone toxicity using postmortem samples. R‐ and S‐methadone were measured in femoral blood from 90 postmortem cases, mainly drug users. The R‐enantiomer concentrations significantly exceeded that of the S‐enantiomers (Wilcoxon’s test, p < 0.001). The samples were divided into four groups according to other drugs detected (methadone only, methadone and strong analgesics, methadone and benzodiazepines, or methadone and other drugs). There was no significant difference in any of the R‐methadone/total methadone ratios among the four groups. The median R/S ratio was 1.38, which tends to be higher than that reported for the plasma of living subjects. In addition, we investigated whether small nucleotide polymorphisms in the MDR1 gene that encode the drug transporter P‐glycoprotein were associated with the concentrations of R‐ and S‐methadone and its metabolite 2‐ethylidene‐1,5‐dimethyl‐3,3‐diphenylpyrrolidine. No significant association was detected.

Dominance of pre-analytical over analytical variation for measurement of methadone and its main metabolite in postmortem femoral blood

On the basis of simultaneously sampled postmortem blood specimens from the left and right femoral veins the pre-analytical variation of methadone measurements was evaluated and compared to the analytical variation. The material consisted of a series of 27 duplicate samples from routine autopsy cases comprising mainly drug addicts. A chiral LC-MS/MS method was used for measurement of the R- and S-enantiomers of methadone and its main metabolite 2-ethyl-1,5-dimethyl-3,3-diphenylpyrrolinium (EDDP). The analytical CV% was determined to be in the range 3-4% for methadone enantiomers and 4-6% for EDDP enantiomers. The total measurement uncertainty (CV(T)) was estimated from the pre-analytical variation (CV(PA)), analytical variation proper (CV(A)), and variation related to calibration (traceability) (CV(Cal)) according to the relationship CV(T) = [CV(2)(PA) + CV(2)(A) + CV(2)(cal)](0.5). Uncertainty related to calibration concerned a component related to the purity of drug reference compound and a contribution from the production of calibrator solutions (CV(Cal)<1%). Pre-analytical sampling variation was estimated from the duplicate measurements of blood samples after subtraction of the analytical component. The pre-analytical variation amounted to a CV% of 19-21% for R- and S-methadone and 30-38% for R- and S-EDDP, i.e. considerably larger than the other components. Due to the squared addition principle, the resulting total uncertainty (CV(T)) became largely identical to the CV(PA), i.e. 19-21% for R- and S-methadone and 31-38% for R- and S-EDDP enantiomers. Accordingly, CV(T) exceeded CV(A) by a factor 5 or more. Dominance of the pre-analytical component of variation may also be likely for other compounds measured in postmortem blood samples. Thus, the width of the 95%-uncertainty interval (+/-2CV(T)) for a postmortem measurement is largely determined by the pre-analytical component of variation. This should be kept in mind when judging on the uncertainty of postmortem measurement results.

Gene dose effects of GSTM1, GSTT1 and GSTP1 polymorphisms on outcome in childhood acute lymphoblastic leukemia

Children with acute lymphoblastic leukemia (ALL) react very differently to chemotherapy. One explanation for this is inherited genetic variation. The glutathione S-transferase (GST) enzymes inactivate a number of chemotherapeutic drugs administered in childhood ALL therapy. Two multiplexing methods were applied for genotyping the GSTM1 and GSTT1 genes (distinguishing between 0, 1, or 2 gene copies) and the GSTP1 313 A>G polymorphism, simultaneously. A total of 263 childhood ALL patients were genotyped. No gene dose effect on outcome was demonstrated with either GST polymorphisms. Grouping of GSTM1 and GSTT1 into poor (0 or 1 gene copy)—and good metabolizers (at least 2 gene copies)—showed that the poor metabolizers had a trend toward a better outcome (event-free survival =91.8%) compared with the good metabolizers (event-free survival =83.2%). Similarly, in the adjusted analysis the good metabolizers demonstrated a 2.2-fold higher risk trend of experiencing an event (resistant disease or relapse) compared with the poor metabolizers (P=0.066; hazard ratio =2.248; 95% confidence interval, 0.948-5.327). In conclusion, our results suggest that the combined gene dose of GSTM1 and GSTT1 may influence outcome in childhood ALL.

ISO 17025 validation of a next-generation sequencing assay for relationship testing.

The HID‐Ion AmpliSeq™ Identity Panel is a next‐generation sequencing assay with 90 autosomal and 34 Y‐chromosome SNPs that are amplified in one PCR step and subsequently sequenced using the Ion Personal Genome Machine (Ion PGM™) System. This assay was validated for relationship testing in our ISO 17025 accredited laboratory in 2015. Here, the essential parts of the validation report submitted to the Danish Accreditation Fund are presented. A total of 100 unrelated Danes were typed in duplicates and the locus balance, heterozygote balance (Hb) and noise levels were analysed in detail. Two loci were disregarded for casework because genotyping was uncertain. Hb for rs7520386 was skewed and high levels of noise were observed in rs576261. Three general acceptance criteria for analysis of single‐source samples were defined: (i) sequencing depth > 200 reads, (ii) noise level < 3% and (iii) Hb > 0.3. A Python script named SNPonPGM was developed to assist the analyst by highlighting loci that do not fulfil the general acceptance criteria. Furthermore, SNPonPGM has functions that reduce the hands‐on time of the reporting officer to a few minutes per case. Mixtures with DNA from two individuals in a 1:24 ratio were readily identified using the three criteria and the SNPonPGM script.

The role of the glutathione S-transferase genes GSTT1, GSTM1, and GSTP1 in acetaminophen-poisoned patients

The aim of this study was to assess if genetic variants in the glutathione-S-transferase genes GST-T1, M1, and P1 reflect risk factors in acetaminophen (APAP)-poisoned patients assessed by investigation of the relation to prothrombin time (PT), which is a sensitive marker of survival in these patients. A total of 104 APAP-poisoned patients were genotyped for deletion polymorphisms in the GSTT1 and GSTM1 genes and for the GSTP1 Ile105Val polymorphism. We found a borderline association (p = 0.05) between the GSTT1 homozygous deletion genotype and high trough PT (a marker of prognosis in APAP poisoning) compared to carrying two functioning copies of the gene. No significant association was found between any of the GSTM1 and GSTP1 genotypes and PT. The frequency of GSTP1 Val/Val genotypes was significantly lower in the patients than in the background population (p = 0.047). The results suggest that the GSTT1 homozygous deletion genotype may be associated with a better prognosis after APAP poisoning and that carriers of the GSTP1 homozygous variant genotype may have a decreased risk of being APAP poisoned.

High-throughput sequencing of forensic genetic samples using punches of FTA cards with buccal swabs.

Here, we demonstrate that punches from buccal swab samples preserved on FTA cards can be used for high-throughput DNA sequencing, also known as massively parallel sequencing (MPS). We typed 44 reference samples with the HID-Ion AmpliSeq Identity Panel using washed 1.2 mm punches from FTA cards with buccal swabs and compared the results with those obtained with DNA extracted using the EZ1 DNA Investigator Kit. Concordant profiles were obtained for all samples. Our protocol includes simple punch, wash, and PCR steps, reducing cost and hands-on time in the laboratory. Furthermore, it facilitates automation of DNA sequencing.

Pharmacokinetic variability of clarithromycin and differences in CYP3A4 activity in patients with cystic fibrosis

BACKGROUND To investigate the correlation between CYP3A4/5 activity and clarithromycin metabolism, and between CYP3A activity and CYP3A genotype. METHODS This is an open-label, prospective pharmacokinetic study evaluating CYP3A activity using The Erythromycin Breath Test. Eight blood samples were collected within 12h after clarithromycin 500 mg was administered orally. The clarithromycin concentrations were measured by liquid chromatography-tandem mass spectrometry. AUC, Tmax and Cmax were calculated. Selected Single Nucleotide polymorphisms in CYP3A4/5 genes were assessed by PCR and single base extension. RESULTS Twenty-one chronically infected patients were included. An 8-fold variation in the CYP3A4 activity, 10-fold variation in AUC for clarithromycin (median 881 μg/mL × min), and a 16-fold variation in Cmax for clarithromycin (median 3.4 μg/mL) were found. A linear correlation between the CYP3A4-activity and clarithromycin metabolism was demonstrated (P < 0.05). CONCLUSION The large variation in the clarithromycin pharmacokinetics in cystic fibrosis patients may cause treatment failure. The Erythromycin Breath Test could be valuable in identifying cystic fibrosis patients in risk of treatment failure/drug toxicity.